Modified Luciola cruciata luciferase protein

ABSTRACT

A codon optimized and stabilized luciferase gene based upon the sequence of the natural luciferase gene isolated from  Luciola cruciata  (Japanese firefly) and a novel recombinant DNA characterized by incorporating this new gene coding for a novel luciferase into a vector DNA for improved activities in mammalian cells, are disclosed. This new luciferase exhibits long-wavelength light emission, as well as improved thermostability and higher expression levels in mammalian cell systems, compared to native luciferase.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. Ser. No. 12/287,561,filed Oct. 10, 2008 entitled MODIFIED “LUCIOLA CRUCIATA LUCIFERASE GENEAND PROTEIN”, which issued as U.S. Pat. No. 7,723,502 on May 25, 2010.

FIELD OF THE INVENTION

The present invention relates to a novel codon optimized and stabilizedluciferase gene (COS luciferase) derived from Japanese firefly Luciolacruciata luciferase. The invention further relates to production of astabilized luciferase protein using such a modified gene.

BACKGROUND OF THE INVENTION

Bioluminescence in certain organisms via the reaction of luciferin andluciferase is well known in the art. The use of the luciferase enzymehas become highly valuable as a genetic marker gene due to theconvenience, sensitivity and linear range of the luminescence assay.Luciferase has been used in many experimental biological systems in bothprokaryotic and eukaryotic cell culture, transgenic plants and animals,as well as cell-free expression systems.

For example, Japanese Firefly Luciola cruciata luciferase expression canbe monitored as a genetic marker in cell extracts when mixed withsubstrates (D-luciferin, Mg²⁺ ATP, and O₂), and the resultingluminescence measured using a luminescent detection device (containing aphotomultiplier system or equivalent) such as liminometers orscintillation counters without the need of a reagent injection device.The Luciola cruciata luciferase activity can also be detected in livingcells by adding D-luciferin or more membrane permeant analogs such asD-luciferin ethyl ester to the growth medium. This in vivo luminescencerelies on the ability of D-luciferin or more membrane permeant analogsto diffuse through cellular and intracellular organelle membranes and onthe intracellular availability of ATP and O₂ in these cells.

Despite its utility as a reporter, current luciferases isolated fromvarious organisms, including insects and marine organisms are notnecessarily optimized for expression or production in systems that areof most interest to the medical community and experimental molecularbiologists. Accordingly, a need exists for a luciferase nucleic acidmolecule that allows improved protein production in mammalian cells andtissues.

SUMMARY OF THE INVENTION

The present invention describes a novel codon optimized and stabilizedluciferase gene coding for an improved luciferase protein. This newluciferase exhibits long-wavelength light emission, as well as improvedthermostability and higher expression levels in mammalian cell systems,compared to native luciferase. Also described is a method of producing astabilized luciferase protein by inserting a nucleic acid molecule ofthe present invention into an appropriate microorganism via a vector andculturing the microorganism to produce the stabilized luciferaseprotein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cleavage map of recombinant plasmid pDC57 DNA withendonuclease restriction enzymes.

FIG. 2 shows a cleavage map of recombinant plasmid pDC99 DNA withendonuclease restriction enzymes.

FIG. 3 shows a comparison of the native L. cruciata luciferase sequencewith the codon optimised nucleotide sequence of the present invention.

FIG. 4. shows a comparison of Luciferase Expression Levels using thepDC99 vector of the present invention with the Photinus pyralisluciferase vector pSV40-GL3 in mammalian cells.

FIG. 5 shows a comparison of the thermal stability of the modifiedLuciola cruciata luciferase protein of the present invention with thatof the Photinus pyralis wild type protein.

DETAILED DESCRIPTION OF THE INVENTION

The wild-type sequence is known for the luciferase molecule from manydifferent species and numerous modifications to those sequences havebeen described in the art. The present invention describes modificationsto the nucleic acid molecule encoding luciferase in the Japanesefirefly, Luciola cruciata as well as the luciferase protein itself. In aparticular embodiment of the invention, the modified Luciola cruciataluciferase nucleic acid molecule encodes an improved luciferase enzymewhich demonstrates greater thermostability (see FIG. 5 for an analysisof the thermal stability of the codon-optimized and stabilized Luciolacruciata luciferase protein of the present invention versus wild typeprotein at various temperatures) as well as a wavelength shift from blueto red compared to native luciferase.

In another embodiment of the present invention, mRNA transcribed fromthe modified luciferase nucleic acid molecule is more stable inmammalian cells. This leads to enhanced levels of mRNA and results ingreater expression of the luciferase (see FIG. 4 for a comparison ofexpression levels using the pDC99 vector of the present invention withthe Photinus pyralis luciferase vector pSV40-GL3 in mammalian cells). Inanother embodiment, the level of mRNA is preferably increased by 10% to200% over that seen when native sequence is expressed in mammaliancells.

In a particular embodiment of the present invention, the modifiedLuciola cruciata luciferase nucleic acid molecule is altered to removeRNAse cleavage motifs. The wild-type sequence shown in SEQ ID NO:1 hasRNAse cleavage motifs at nucleotides 384-388, 682-686, and 929-933. In apreferred embodiment, the modified sequence is changed as shown in SEQID NO:3 and FIG. 3 to remove these motifs. In particular, nucleotides384 to 388 are changed from (ATTTA) to GTTCA, nucleotides 682 to 686 arechanged from (ATTTA) to ATCTA and nucleotides 929 to 933 are changedfrom ATTTA) to ACCTG.

Vectors such as retroviral vectors or other vectors intended for theintroduction of recombinant DNA into mammalian cells will often containactive splice donor sequences. Instability is often created when a wildtype gene from a non-mammal is carried by a retroviral vector due to therecognition of cryptic splice acceptor sequences in the wild type geneand splicing between these and splice donor sites present in the vector.In a particular embodiment of the present invention, cryptic spliceacceptor sequences present in the wild type L. cruciata luciferasenucleic acid molecule are altered or removed.

In another particular embodiment of the present invention, crypticsplice acceptor sites found at bases 448 to 463, 919 to 934, 924 to 939,940 to 955, 1148 to 1163, 1156 to 1171, 1159 to 1174, and 1171 to 1186of the wild type sequence of SEQ ID NO:1 have one or more nucleotidesaltered.

In a particular embodiment, bases 448 to 463 of the wild type L.cruciata luciferase, i.e. ACCATTGTTATACTAG, herein SEQ ID NO:5 arechanged in the COS luciferase to ACCATCGTGATCCTGG herein SEQ ID NO:6.

In another embodiment, bases 919 to 934 of the wild type L. cruciataluciferase, i.e. GATTTGTCAAATTTAG herein SEQ ID NO:7 are changed in theCOS luciferase to GACCTGAGCAACCTGG herein SEQ ID NO:8.

In another embodiment, bases 924 to 939 of the wild type L. cruciataluciferase, i.e. GTCAAATTTAGTTGAG herein SEQ ID NO:9 are changed in theCOS luciferase to GAGCAACCTGGTGGAG herein SEQ ID NO:10.

In another embodiment, bases 940 to 955 of the wild type L. cruciataluciferase, i.e. ATTGCATCTGGCGGAG herein SEQ ID NO:11 are changed in theCOS luciferase to ATCGCCAGCGGCCGGAG herein SEQ ID NO:12.

In another embodiment, bases 1148 to 1163 of the wild type L. cruciataluciferase, i.e. CTTTAGGTCCTAACAG herein SEQ ID NO:13 are changed in theCOS luciferase to GCCATCATCATCACC herein SEQ ID NO:14.

In another embodiment, bases 11.56 to 1171 of the wild type L. cruciataluciferase, i.e. CCTAACAGACGTGGAG herein SEQ ID NO: 15 are changed inthe COS luciferase to ATCACCCCCGAGGGCG herein SEQ ID NO:16.

In another embodiment, bases 1159 to 1174 of the wild type L. cruciataluciferase, i.e. AACAGACGTGGAGAAG herein SEQ ID NO:17 are changed in theCOS luciferase to AACAGACGGGGCGAAG herein SEQ ID NO:18.

In another embodiment, bases 1171 to 1186 of the wild type L. cruciataluciferase, i.e. GAAGTTTGTGTTAAAG herein SEQ ID NO:19 are changed in theCOS luciferase to CGACGACAAGCCTGGA herein SEQ ID NO:20.

In a particular embodiment, the corresponding branchpoint sequences forthe above cryptic splice sites in the wild type L. cruciata luciferaseSEQ ID NO:1, are also altered to further suppress the splicingpotential.

Palindromic sequences tend to have an adverse effect on translationalefficiency and/or mRNA stability. The degree of these effects aregenerally directly related to the stability of the loop structuresformed by these palindromic motifs. Accordingly, one embodiment of thepresent invention includes reducing the number of palindromic motifs. Ina particular embodiment, palindromic motifs are altered by one or morenucleotides without altering the encoded luciferase enzyme activity andpreferably without altering the amino acid sequence.

In a particular embodiment, a palindromic pair of motifs at bases 1087to 1095 and 1218 to 1226 of the wild type L. cruciata luciferase, ie.GCTTCTGGA and TCCAGAAGC, respectively are changed in the COS luciferaseto GCCAGCGGC and CCCCGAGGC, respectively.

In a particular embodiment, a palindromic pair of motifs at bases 1151to 1158 and 1185 to 1192 of the wild type L. cruciata luciferase, ie.TAGGTCCT and AGGACCTA, respectively are changed in the COS luciferase toTGGGCCCC and GGGCCCCA, respectively.

In a particular embodiment, a palindromic pair of motifs at bases 255 to264 and 350 to 359 of the wild type L. cruciata luciferase, ie.AAACTGTGAA and TTCACAGTTT, herein SEQ ID NO: 21 and SEQ ID NO:22respectively are changed in the COS luciferase to GAACTGCGAG andTGCACAGCCT herein SEQ ID NO:23 and SEQ ID NO:24, respectively.

In a particular embodiment, a palindromic pair of motifs at bases 1381to 1389 and 1508 to 1516 of the wild type L. cruciata luciferase, ie.TTGCAACAT and ATGTTGCAA, respectively are changed in the COS luciferaseto CTGCAGCAC and ACGTCGCCA, respectively.

In a particular embodiment, a palindromic pair of motifs at bases 235 to242 and 883 to 890 of the wild type L. cruciata luciferase, ie. AGAATTGCand GCAATTCT, respectively are changed in the COS luciferase to CGGATCGCand GCCATCCT, respectively.

In a particular embodiment, a palindromic pair of motifs at bases 445 to452 and 740 to 747 of the wild type L. cruciata luciferase, ie. AAAACCATand ATGGTTTT, respectively are changed in the COS luciferase to AAGACCATand ACGGCTTC, respectively.

The wild type Luciola cruciata sequence incorporates several negativelycis-acting motifs that hamper expression in mammals are found in thewild-type sequence. In a particular embodiment of the present invention,the modified sequence contains no negative cis-acting sites (such assplice sites, poly(A) signals, etc.) which would negatively influenceexpression in mammalian cells.

The wild type Luciola cruciata sequence has a GC content that is quitelow compared to mammalian sequences, which facilitates quick mRNAturnover. In another embodiment, the GC-content of the modifiedluciferase sequence is increased from about 37% to about 62%, prolongingmRNA half-life. Codon usage was adapted to the bias of Homo sapiensresulting in a high CAI (codon adaptation index) value of 0.97, incomparison to 0.62 for the wild-type sequence. Accordingly, theoptimized gene provides high and stable expression rates in Homo sapiensor other mammalian cell types.

The codon usage alterations generally lead to an increase thetranslation efficiency of the messenger RNA in a mammalian cell. It is afeature of the present invention that mRNA transcribed from the modifiedluciferase gene is more stably present in mammalian cells. This leads toenhanced levels of mRNA and results in greater expression of theluciferase protein. In a particular embodiment of the present invention,the level of mRNA is increased by 10% to 200% compared to expression ofthe native gene in the same cell. The codon optimization modificationsare preferably incorporated such that resulting modified enzyme activityis not altered and most preferably that the amino acid sequence is notaltered, except for desired changes described herein.

Many organisms display a bias for use of particular codons to code foraddition of a specific amino acid in a growing peptide chain. Codonbiases for differences in codon image between organisms often correlatewith the efficiency of translation of messenger RNA (mRNA), which inturn is believed to result from the properties of the codons beingtranslated and the availability of particular transfer RNA (tRNA)molecules to be used in translation of the mRNA into protein. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis.

Codon usage in highly expressed mammalian genes are as follows:

[AminoAcid Codon Fraction] Gly GGG 0.15 Gly GGA 0.18 Gly GGT 0.21 GlyGGC 0.46 Glu GAG 0.68 Glu GAA 0.32 Asp GAT 0.38 Asp GAC 0.62 Val GTG0.54 Val GTA 0.08 Val GTT 0.14 Val GTC 0.25 Ala GCG 0.14 Ala GCA 0.13Ala GCT 0.29 Ala GCC 0.44 Arg AGG 0.14 Arg AGA 0.13 Ser AGT 0.10 Ser AGC0.25 Lys AAG 0.75 Lys AAA 0.25 Asn AAT 0.26 Asn AAC 0.74 Met ATG 1. 00Ile ATA 0.06 Ile ATT 0.26 Ile ATC 0.67 Thr ACG 0.09 Thr ACA 0.18 Thr ACT0.23 Thr ACC 0.50 Trp TGG 1. 00 End TGA 0.30 Cys TGT 0.46 Cys TGC 0.54End TAG 0.16 End TAA 0.53 Tyr TAT 0.35 Tyr TAC 0.65 Leu TTG 0.10 Leu TTA0.03 Phe TTT 0.35 Phe TTC 0.65 Ser TCG 0.07 Ser TCA 0.08 Ser TCT 0.20Ser TCC 0.31 Arg CGG 0.11 Arg CGA 0.05 Arg CGT 0.17 Arg CGC 0.40 Gin CAG0.82 Gln CAA 0.18 His CAT 0.35 His CAC 0.65 Leu CTG 0.56 Leu CTA 0.05Leu CTT 0.08 Leu CTC 0.18 Pro CCG 0.16 Pro CCA 0.19 Pro CCT 0.30 Pro CCC0.35 The codon bias in the Gene is different to the highly expressedmammalian genes. Of the codons that potentially encode a particularamino some are very rarely used.

By the standard set forth in the preceding paragraph, the wild typeLuciola cruciata sequence uses codons rarely used in mammalian systemswith a high frequency. To have the most impact the most rarely usedcodons in highly expressed mammalian genes are preferably changed. Inone embodiment of the present invention, at least about 90% of therarely used codons found in the wild type sequence are altered to morepreferred codons for the corresponding amino acid.

For example, the codon TTA is used to encode leucine in only 3% of casesin highly expressed mammalian systems, but is seen in the wild typeluciferase of SEQ ID NO:1 at positions 87-89, 246-248, 339-341, 360-362,405-407, 720-722, 774-776, 801-803, 828-830, 906-908, 933-935, 963-965,1032-1034, 1152-1154, 1329-1331, 1368-1370, and 1542-1544. In oneembodiment of the present invention, each of these positions is changedto CTG, which is a more commonly used in mammalian systems, thusoptimizing the nucleic acid sequence for expression in mammals withoutchanging the amino acid sequence. A preferred altered Luciola cruciataLuciferase gene is one where at least about 70%, 80%, 90%, 95%, 99% or100% of codons are thus optimized for expression in a particular cellsystem. A specific embodiment of the present invention is the codonoptimized and stabilized (COS) Luciferase set forth in SEQ ID NO:3

In another embodiment of the present invention, it is anticipated thatconservative amino acid substitutions might be made throughout theenzyme without adversely altering the enzyme activity. One or more aminoacid residues within the sequence can be substituted by another aminoacid of a similar polarity, which acts as a functional equivalent,resulting in a silent alteration.

Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, praline, phenylalanine, tryptophan and methionine.Amino acids containing aromatic ring structures are phenylalanine,tryptophan, and tyrosine. The polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Thepositively charged (basic) amino acids include arginine, lysine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid.

Particularly conservative amino acid substitutions are: (a) Lys for Argor vice versa such that a positive charge may be maintained; (b) Glu forAsp or vice versa such that a negative charge may be maintained; (c) Serfor Thr or vice versa such that a free OH can be maintained; (d) Gln forAsn or vice versa such that a free NH₂ can be maintained; (e) Ile forLeu or for Val or vice versa as roughly equivalent hydrophobic aminoacids; and (f) Phe for Tyr or vice versa as roughly equivalent aromaticamino acids. However it will be understood that less conservativesubstitutions may still be made without affecting the activity of theresulting luciferase enzyme.

In a particular embodiment of the present invention, the amino acidencoded at nucleotide position 875-877 in the wild-type sequence, SEQ IDNO:1 is changed from Ser(S) to Tyr(Y). This nucleic position correspondswith position 286 of the wild-type protein sequence, SEQ ID NO:2. Thismodification was found to have the surprising effect of making theresulting protein >100-fold more stable after 1 hour and >1000-fold morestable after 2 hours at 37° C. The modified luciferase also demonstratedgreater thermostability than wild-type protein at room temperature. Thesubstitution of Tyr for Ser at this position was also shown to have thesurprising effect of shifting the emitted light from blue to red (from582 nm to 619 nm (pH 6)). The present invention also anticipates similarconservative amino acid substitutions at nucleotide position 875-877,including substituting Tyr, Lys, Leu, or Gln for Ser.

It will be understood that the invention also encompasses a method ofusing the modified luciferase gene as a marker gene in live cells,wherein the nucleic acid molecules encoding the modified luciferase geneare provided in an expression vector with appropriate cis- andtrans-acting expression elements and thereby provide cells expressingthe modified luciferase gene that produce the modified enzymeintracellularly.

The modified luciferase of the present invention might be incorporatedas part of a fusion protein. Additionally the invention encompasses acloning vehicle having a sequence encoding the modified luciferase gene.

The luciferase gene will typically be positioned operably linked to apromoter. Preferably the promoter is a mammalian promoter, and may beselected from one of the many known mammalian promoters. In the contextof this invention the term luciferase gene refers to the open readingframe encoding the modified luciferase protein.

Additionally other nucleotide motifs might be introduced to enhancetranscription and/or translation such as a Kozak consensus sequence ortranscriptional enhancers.

The present invention describes a plasmid vector for expression inmammalian cells, a bacterial vector for expression in plant cells, butalso contemplates a retroviral vector or a lentiviral vector, thatincludes the modified luciferase gene, or a cell carrying the modifiedluciferase gene.

The examples below are given so as to illustrate the practice of thisinvention. They are not intended to limit or define the entire scope ofthis invention.

EXAMPLES Example 1

Construction of the modified Luciola cruciata Luciferase Gene. Thesynthetic COS luciferase gene, SEQ ID NO:3, was assembled from syntheticoligonucleotides and/or PCR products. The fragment was cloned into pMK(kanR) using. KpnI and Sad restriction sites. The plasmid DNA waspurified (Pure Yield™ Plasmid Midiprep, Promega) from transformedbacteria and concentration determined by UV spectroscopy. The finalconstruct was verified by sequencing. The sequence congruence within theused restriction sites was 100%.

Example 2

Subcloning of the modified Luciola cruciata Luciferase Gene into thepCMV and pSV40 vectors.

The synthetic COS luciferase assembled in Example 1 was excised from pMKcloning vector using flanking XhoI and NotI restriction enzymes (FastDigest, Fermentas). The excised fragment was gel-purified (GenElute GelExtraction Kit, Sigma) and quantitated using MassRuler™ DNA Ladder Mix(Fermentas). The excised gene was subcloned into both pCMV and pSV40Mammalian Expression Vectors using corresponding XhoI and NotIrestriction sites. The completed pCMV construct was named pDC57. Thecompleted pSV40 construct was named pDC99.

Example 3

Subcloning of the modified Luciola cruciata Luciferase Gene into thepNosdc binary vector for expression in plants.

The synthetic COS luciferase assembled in Example 1 was amplified usingthe Polymerase Chain reaction. Amplification was performed with primersincluding XmaI and Sad restriction sites. The ends of the amplifiedfragment were cut with XmaI and Sad restriction enzymes (New EnglandBiolabs) and the fragment was gel-purified (GenElute Gel Extraction Kit,Sigma) and quantitated using MassRuler™ DNA Ladder Mix (Fermentas). Theamplified fragment was subcloned into the pNosdc binary vector fortransformation of plants via Agrobacterium tumefaciens. The completedconstruct was named pNosdcCOS.

Example 4

Transfection of Mammalian Cells with the modified Luciola cruciataLuciferase vectors pDC57 and pDC99.

NIH 3T3 cells (murine tumor fibroblasts) were grown to 80% confluence in100 mm tissue culture plates. Cells were transfected with either pDC57or pDC99 using Lipofectamine and PLUS reagents (Invitrogen).

Example 5

Analysis of Luciferase Expression Levels using the pDC99 Vector andComparison to the Luciferase Expression Using the Photinus pyralisluciferase Vector pSV40-GL3 in mammalian cells.

Transfected NIH 3T3 cells prepared in Example 4 were lysed using a lysisbuffer comprised of 25 mM Tris-phosphate (pH 7.8), containing 10%glycerol, 1% Triton X-100, 1 mg/ml BSA, 2 mM EGTA and 2 mM DTT. Cellswere washed with 1× Phosphate Buffered Saline, and lysis buffer (1 mL)was added to surface of plate. Plate was incubated for 30 mins, andlysate was collected. Additionally, NIH 3T3 cells were transfected withpSV40-GL3, a construct containing wild type luciferase from Photinuspyralis, as per the method in Example 4 and lysed using the abovemethod. As a negative control, untransfected NIH 3T3 cells were alsolysed by the above method.

Cell lysates were diluted using lysis buffer, and added in triplicate towells of a solid white 96-well plate (Costar). Added to cell lysates wasa reagent containing 1 mM D-luciferin and 2 mM ATP in a buffer comprisedof 25 mM Glycylglycine, 15 mM MgSO4, 4 mM EDTA, 15 mM Potassiumphosphate pH 7.8, 1 mM DTT, and 1 mM Coenzyme A.

Luminescence was recorded using a Perkin-Elmer HTS7000 Plus Bio AssayReader (200 ms integration time). Results of these analyses are shown inFIG. 4.

Example 6

Analysis of the thermal stability of the modified Luciola cruciataLuciferase Protein versus wild type protein.

Cell lysates from NIH 3T3 cells transfected with pDC99 and pSV40-GL3(transfected according to method in Example 4), as well as untransfectedcells were prepared as described in Example 5a. Luminescence of eachsample was recorded as described in Example 5a to obtain a baselinevalue of enzyme activity. Portions of each sample were then incubated inwater baths at 37° C., 42° C., and 55° C. A portion of each sample wasalso incubated at ambient room temperature (25° C.). At 1 hour and 2hour intervals, aliquots of each temperature-incubation were removed andassayed for activity using the method described in Example 5a. Resultsof these analyses are shown in FIG. 5.

Example 7

Isolation of the modified Luciola cruciata Luciferase Protein frombacterial culture.

Escherichia coli (strain JM109) harboring a plasmid vector containing aHistidine tag (such as pDEST17 (Invitrogen), pET-14b (Novagen and pQE(Qiagen)) fused to the codon optimized and stabilized luciferase gene(COS) are grown to an OD600 of 0.2 by incubation at 37° C. with vigorousshaking in 250 mL LB Broth containing the appropriate selectionantibiotic. Bacterial cells are pelleted by centrifugation at 5,000×g,and the pellet is resuspended in a bacterial cell lysis buffer, such asCellLytic B (Sigma Prod. No. B7435). The suspension is incubated for 15rains to extract soluble proteins, and then centrifuged at >15,000×g for10 mins to pellet insoluble debris. The lysate is applied to an affinitycolumn (such as HIS-Select, Sigma Prod. No. H7787) equilibrated with0.1M sodium phosphate, 8M urea, pH 8.0 (equilibration buffer).Impurities are removed by washing the column several times withequilibration buffer. The His-tagged COS protein is eluted from thecolumn using an acidic buffer, such as 0.1 M sodium phosphate, 8 M urea,with a pH in the 4.5-6.0 range. The eluate contains the recombinantcodon optimized and stabilized luciferase protein. The purified proteinis then dialyzed against H2O and lyophilized. The lyophilized protein isdissolved in reaction buffer (25 mM Glycylglycine, 15 mM MgSO4, 4 mMEDTA, 15 mM Potassium phosphate pH 7.8, 1 mM DTT, and 1 mM Coenzyme A)or dH₂O and assayed by adding a reagent containing 1 mM D-luciferin and2 mM ATP in reaction buffer.

Luminescence is recorded using a Perkin-Elmer HTS7000 Plus Bio AssayReader (200 ms integration time).

Example 8

Transfection of plants with codon optimized and stabilized luciferase(COS).

Agrobacterium tumefaciens are transfected with pdcNosCOS according tofreeze-thaw protocol previously described (D. Weigel, J. Glazerbrook,pp. 125-126 (2002)). Arabidopsis thaliana (strain CS-20) are transfectedby the floral dip method using the aforementioned transfectedAgrobacterium, using the protocol described previously (D. Weigel, J.Glazerbrook, pp. 129-130 (2002)). Seedlings are selected on Murashigeand Skoog Agar plates containing 50 μg/mL kanamycin, as describedpreviously (D. Weigel, J. Glazerbrook, pp. 131-132 (2002)).

Protein is extracted from plant tissue according to the followingprocedure: Tissue is lyophilized and ground into a fine powder in amortar. The powder is placed in a microcentrifuge tube and suspended inreaction buffer (25 mM Glycylglycine, 15 mM MgSO4, 4 mM EDTA, 15 mMPotassium phosphate pH 7.8, 1 mM DTT, and 1 mM Coenzyme A) by vortexing.The tube is incubated at 10 mins at room temperature to solubilizeproteins, followed by centrifugation at >15,000×g to pellet solidmaterial. The supernatant is transferred to a fresh tube, and added intriplicate to wells of a solid white 96-well plate (Costar). Added totissue extracts is a reagent containing 1 mM D-luciferin and 2 mM ATP ina buffer comprised of 25 mM Glycylglycine, 15 mM MgSO4, 4 mM EDTA, 15 mMPotassium phosphate pH 7.8, 1 mM DTT, and 1 mM Coenzyme A.

Luminescence is recorded using a Perkin-Elmer HTS7000 Plus Bio AssayReader (200 ms integration time).

It is to be understood that, while the foregoing invention has beendescribed in detail by way of illustration and example, numerousmodifications, substitutions, and alterations are possible withoutdeparting from the spirit and scope of the invention as described in thefollowing claims.

1. An isolated protein encoded by a nucleic acid molecule comprising the nucleic acid sequence as set forth in SEQ ID NO:3.
 2. The isolated protein of claim 1, wherein said protein comprises the amino acid sequence as set forth in SEQ ID NO:4.
 3. The isolated protein of claim 1, wherein said protein is produced by a method comprising culturing, in a medium, a microorganism belonging to the genus Escherichia having inserted therein a nucleic acid sequence encoding said protein and collecting the luciferase protein from the culture.
 4. The isolated protein of claim 1, wherein said protein exhibits bioluminescence in the presence of D-luciferin at a wavelength between about 590 nm and about 620 nm.
 5. A kit for detecting ATP comprising: A. the isolated protein of claim 1; and B. at least one ATPase inhibitor, wherein the combination of said protein and said ATPase inhibitor forms a composition for detecting ATP in a sample.
 6. An isolated amino acid molecule comprising the amino acid sequence as set forth in SEQ ID NO:4. 